Abstract: BiTE ® (Bispecific T-cell Engager) constructs represent a novel immunotherapeutic strategy that recruits T cells against cancer cells independent of their TCR specificity. Currently, two CD33xCD3 BiTE ® antibody constructs (AMG 330 & AMG 673) are being investigated in phase I dose escalation trials in patients with relapsed/refractory Acute Myeloid Leukemia (AML) with early evidence of acceptable safety and anti-leukemic activity (Ravandi et al., ASH 2020; Subklewe et al., EHA 2020). So far, details of BiTE ® mediated T-cell engagement and information on parameters contributing to their efficacy need more investigation. Therefore, we aimed to characterize the interplay between target and effector cells to deepen our mechanistic understanding of BiTE ® construct mediated T-cell engagement. Previously, we have created a novel in vitro model system with murine Ba/F3 cells expressing human (hu) CD33 ± huCD80 ± huCD86 ± huPD-L1 to study T-cell proliferation and cytotoxicity induced by AMG 330. Using that system, we showed that expression of T-cell co-signaling receptors on target cells modulate AMG 330 induced T-cell activity (Marcinek et al., ASH 2018, EHA 2019). Here, we hypothesize that expression of costimulatory molecules impacts BiTE ® mediated immune synapse formation and consecutive downstream signaling in BiTE ® construct activated T cells. To study whether AMG 330 can induce synapse formation and TCR triggering we used a previously described reconstituted T-cell system, which consists of non-immune (HEK) cells introduced with genes encoding the TCR and other proteins (e.g. CD45) required for the regulation of TCR phosphorylation (James et al., Nature 2012). HEK-T cells were incubated with huCD33 transduced RajiB cells in presence of fluorescently labeled AMG 330 or a control BiTE® (cBiTE) construct to allow cell conjugation. A spinning disc confocal microscope system was used to image cells. To pinpoint the role of T-cell co-signaling receptors in immune synapse formation we incubated differentBa/F3 cell constructs or primary AML (pAML) cells with healthy donor T cells in the presence of AMG 330 and analyzed intensity of LFA-1 expression within the synapse using an Imaging Flow Cytometer. Furthermore, we determined phosphorylation of ZAP70, AKT and ERK in conjugated T cells after various time points by phosphoflow cytometry. We observed that AMG 330, in contrast to cBiTE®, induced TCR triggering reflected by exclusion of CD45 from the RajiB-T-cell-interface. Simultaneously clustering of CD33 occurred in AMG 330 induced cell-cell-interfaces (Fig. 1A/B). The percentage of conjugates formed with huCD33 + Ba/F3 cells was significantly higher in constructs expressing huCD86, compared to those expressing no costimulatory antigens or additional huPD-L1 (Mean % in huCD33 + Ba/F3: 2.8 vs. huCD33 + CD86 +.Ba/F3: 4.2 [p=0.0031] vs. huCD33 + huCD86 + PD-L1 + Ba/F3: 2.8 [p=0.0018]). This was accompanied by LFA-1 accumulation within the T-cell-Ba/F3 cell synapse (Mean of MFI in huCD33 + CD86 +.Ba/F3: 10,933 & gt; huCD33 + huCD86 + PD-L1 + Ba/F3: 7,749 & gt; huCD33 + Ba/F3: 7,028). For downstream signaling in T cells after engagement with Ba/F3 cell constructs in the presence of AMG 330, we observed that kinase phosphorylation was highest after 10 minutes in CD86 co-expressing Ba/F3 cells (Mean % of phosphorylation in T-cell conjugates with huCD33 + vs huCD33 + huCD86 + vs huCD33 + CD86 +.PD-L1 + Ba/F3: pERK 40.9 vs 54.3 [p=0.0064] vs 51.2 %; pAKT: 69.1 vs 81.5 [p=0.0642] vs 75.1 %; pZAP70: 6.9 vs 12.2 [p & lt;0.0001] vs 7.7 % [p & lt;0.0001]) (Fig. 1C). Finally, we evaluated if these finding could also be observed in pAML samples. For that, we determined LFA-1 expression intensity within AMG 330-induced pAML-T-cell synapses. We used CD33 + pAML samples with either high CD86 and no PD-L1 expression or vice versa. Comparing synapse formation between these samples, LFA-1 intensity was 4.6-fold higher in the CD86 + PD-L1 - sample compared to the CD86 - PD-L1 + pAML. Taken together, our data unravel molecular mechanisms of BiTE® construct induced immune synapse formation, highlighting the role of costimulatory molecules in this process. They support the notion that T cell co-signaling receptors like CD86 and PD-L1 modulate T-cell response in an early event manner. Prospective analyses in clinical trials are needed to validate the relevance of checkpoint molecule expression on target cells as a potential predictive biomarker for response. Figure 1 Figure 1. Disclosures Brauchle: Adivo: Current Employment. Lacher: Roche: Research Funding. Kischel: Amgen GmbH Munich: Current Employment. von Bergwelt: Roche: Honoraria, Research Funding, Speakers Bureau; Miltenyi: Honoraria, Research Funding, Speakers Bureau; Mologen: Honoraria, Research Funding, Speakers Bureau; Kite/Gilead: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Astellas: Honoraria, Research Funding, Speakers Bureau; MSD Sharpe & Dohme: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau. Theurich: Amgen: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; GSK: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Takeda: Consultancy, Honoraria. Buecklein: Novartis: Consultancy, Other: congress and travel support, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau; Miltenyi: Research Funding; Kite/Gilead: Consultancy, Honoraria, Other: Congress and travel support, Research Funding; BMS/Celgene: Consultancy, Research Funding; Amgen: Consultancy, Honoraria. Subklewe: Janssen: Consultancy; Seattle Genetics: Consultancy, Research Funding; Roche: Research Funding; Novartis: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Klinikum der Universität München: Current Employment; Takeda: Speakers Bureau; MorphoSys: Research Funding; Miltenyi: Research Funding; Gilead: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; BMS/Celgene: Consultancy, Research Funding, Speakers Bureau.

Abstract: In the past decade outcome of adult ALL was improved significantly using pediatric-based chemotherapy in combination with risk-adapted stem cell transplantation (SCT) and targeted therapies. The GMALL Trial 08/2013 (NCT02881086) for patients (pts) aged 18-55 years (yrs) with newly diagnosed ALL/LBL has a BFM-based 2-phase induction, up to 8 cycles of PEG-asparaginase (ASP) with up to 7 cycles of HDMTX, HDAC, a reinduction phase and conventional maintenance up to 2.5 yrs. Two cycles Nelarabine are implemented for standard risk (SR) T-ALL. Pts with B-precursor-ALL (B-ALL) receive Rituximab independent of CD20 expression (Ph+ only if CD20+). Ph+ ALL pts receive Imatinib and a dose reduced induction (Vincristine, Dexamethasone, ASP). ASP is scheduled in induction 1 (IP1) (d 20) and 2 (d 34 for Ph+ and d 44 for Ph-) and dose is adapted to risk factors for hepatotoxicity. Pts with BMI & gt;30 and/or liver steatosis receive 500 U/m 2, whereas 2000 U/m 2 is the standard dose. It is recommended to withhold the 2 nd dose of ASP in case of clinically relevant ASP-associated toxicities. Pts with high-risk (HR) features (WBC & gt; 30,000/µl in B-ALL, pro B-ALL-, KMT2A rearrangement, early/mature T-ALL) or Ph+ ALL are considered for SCT in CR1 after 1 st consolidation (C1). Pts with MolFail (table 1) after C1 are candidates for targeted therapy (Blinatumomab, Nelarabin) followed by SCT. Randomization (R) I evaluates CNS irradiation versus i.th. prophylaxis in B- ALL/LBL. R II compares SCT versus SR therapy in HR pts with MolCR after induction. Both randomizations are blinded and not available for analysis. The trial is ongoing and scheduled to recruit 950 pts. Between 8/2017-4/2021 770 pts from 78 centers were included and 705 were evaluable. The median age was 35 (18-55) yrs, 638 had ALL (B,Ph-: 55%, Ph+: 20%, T: 25%,) and 67 pts LBL (B:12%; T:88%). For ALL the hematologic (Hem) CR rate after C1 was 93% and the MolCR Rate 61% (75% mol. response) (table 1). HemCR rate was 72% in LBL with 21% PR. PET CT was negative in half of the LBL PR cases. The lowest HemCR rate was observed in early T-ALL (83%) together with a MolCR rate of 45% (71% mol. response). In Ph- pts 2000 U/m 2 as first dose ASP was administered in 66%, 500 U/m 2 in 24%, no ASP in 1% and other doses in 9%. In pts with Ph+ ALL the respective numbers were 61%, 21%, 6% and 13%. For the 2nd dose the numbers were 43%, 21%,13% and 22% for Ph- and 41%, 24%, 13% and 22% for Ph+ pts. Bilirubine increases grade III/IV were observed in Ph- ALL in 18% of the cases treated with 500 U/m 2 vs 24% for 2000 U/m 2. In Ph+ ALL the respective numbers were 5% vs 3%. GOT/GPT grade III/IV increases were observed 29% of Ph- ALL pts treated with 500 U/m 2 vs 25% of those with 2000 U/m 2. The respective numbers for Ph+ ALL were 24% and 40%. At a median follow-up of 23 mo, the overall survival (OS) for all pts (N=705) was 88% and 76% at 1 and 3 yrs resp (subgroups see table 1). OS was correlated to age, 87%, 74%, 69% and 73% at 3 yrs for pts aged 18-25, 26-35, 36-45 and 46-55 yrs resp. SR T-ALL reached an OS of 86% at 3 yrs with a conventional and nelarabine based consolidation. 79% of pts with an indication for SCT were transplanted (64 sibling, 174 MUD). The OS of SCT pts after SCT was 75% at 3 yrs. 63 pts with MolFail became candidates for a targeted therapy, which was realized in 89% of the cases. The molecular response was evaluable in 51 pts and reached 55% (N=40) and 18% (N=11) after one cycle of Blinatumomab or Nelarabin resp. Pts with MolFail (N=63) achieved an OS of 84% at 1y and 72% at 3 years resp (71% for Ph- and 76% for Ph+). This large, ongoing prospective multicenter trial provides promising preliminary results. This applies also to those aged 45-55 yrs underlining that adult pts beyond the variable AYA definitions can benefit from pediatric-based therapy. Intensive and individualized ASP therapy was feasible. Whereas high HemCR rates were observed in nearly all subgroups, MolCR rates ranged from 41-74% and a relevant proportion remained MRD low positive underlining the need for detailed MRD classification. MRD-based targeted treatment was realized in a high proportion of pts. Responses to Blinatumomab in MolFail were lower compared to previous trials. Responses to Nelarabine in MolFail T-ALL pts were only 18%. OS of MolFail pts however was promising with the combination of targeted therapy and SCT. Thus, SCT is still a key component, contributing to improved outcomes although the overall proportion of SCT was lower than in previous GMALL trials. Funded by Deutsche Krebshilfe Figure 1 Figure 1. Disclosures Goekbuget: Servier: Consultancy, Other; Morphosys: Consultancy; Astra Zeneca: Other: Invited talk for company sponsored symposia (with honor); Amgen: Consultancy, Other: Invited talks for company sponsored symposia (with honoraria); Research funding for institution; Novartis: Consultancy, Other: Research funding for Institution; Pfizer: Consultancy, Other: Research funding for institution; Jazz Pharmaceuticals: Other: Research funding for institution; Incyte: Other: Research funding for Institution; Cellestia: Consultancy; Erytech: Consultancy; Abbvie: Other; Gilead/Kite: Consultancy. Stelljes: Pfizer: Consultancy, Research Funding, Speakers Bureau; Medac: Speakers Bureau; Amgen: Consultancy, Speakers Bureau; MSD: Consultancy, Speakers Bureau; Celgene/BMS: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Kite/Gilead: Consultancy, Speakers Bureau. Viardot: Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; University Hospital of Ulm: Current Employment; F. Hoffmann-La Roche Ltd: Honoraria, Membership on an entity's Board of Directors or advisory committees; Kite/Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Nachtkamp: Jazz: Honoraria; Bsh medical: Honoraria; Celgene: Other: Travel Support. Topp: Novartis: Consultancy; Roche: Consultancy, Research Funding; Kite, a Gilead Company: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy; Amgen: Consultancy, Research Funding; Macrogeniecs: Research Funding; Regeneron: Consultancy, Research Funding; Gilead: Research Funding; Universitatklinikum Wurzburg: Current Employment. Wäsch: Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Pfizer: Consultancy; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Novartis: Consultancy; BMS/Celgene: Consultancy; Gilead: Consultancy. Vucinic: Abbvie: Honoraria, Other: Travel Sponsoring; MSD: Honoraria; Janssen: Honoraria, Other: Travel Sponsoring; Gilead: Honoraria, Other: Travel Sponsoring; Novartis: Honoraria. Baldus: Novartis: Honoraria; Amgen: Honoraria; Celgene/BMS: Honoraria; Jazz: Honoraria. Brüggemann: Amgen: Other: Advisory Board, Travel support, Research Funding, Speakers Bureau; Incyte: Other: Advisory Board; Janssen: Speakers Bureau. Pfeifer: Incyte: Honoraria, Research Funding. Schwartz: Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Travel grants, Speakers Bureau; Novartis: Speakers Bureau; Jazz Pharmaceuticals: Other: Travel grants, Speakers Bureau; BTG International Inc: Membership on an entity's Board of Directors or advisory committees; MSD Sharp & Dohme: Membership on an entity's Board of Directors or advisory committees; Basilea: Other: Travel grants; Gilead: Other: Travel grants, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Morphosys: Research Funding. Fiedler: Abbvie: Consultancy, Honoraria, Other: Meeting attendance, Preparation of information material; Amgen: Consultancy, Honoraria, Other: Meeting attendance, Preparation of information material, Patents & Royalties, Research Funding; Ariad/Incyte: Honoraria; Celgene: Consultancy, Honoraria; Jazz: Consultancy, Honoraria, Other: Meeting attendance, Preparation of information material; MorphoSys: Consultancy, Honoraria; Novartis: Honoraria; Pfizer: Consultancy, Honoraria, Research Funding; Daiichi Sanyko: Consultancy, Other: Meeting attendance, Preparation of information material; Stemline: Consultancy; Servier: Consultancy, Other: Meeting attendance, Preparation of information material. OffLabel Disclosure: Nelarabine in newly diagnosed T-ALL

Abstract: Prediction of resistant disease at initial diagnosis of acute myeloid leukemia (AML) can be achieved with high accuracy using cytogenetic data and 29 gene expression markers (Predictive Score 29 Medical Research Council; PS29MRC). Our aim was to establish PS29MRC as a clinically usable assay by using the widely implemented NanoString platform and further validate the classifier in a more recently treated patient cohort. Analyses were performed on 351 patients with newly diagnosed AML intensively treated within the German AML Cooperative Group registry. As a continuous variable, PS29MRC performed best in predicting induction failure in comparison with previously published risk models. The classifier was strongly associated with overall survival. We were able to establish a previously defined cutoff that allows classifier dichotomization (PS29MRCdic). PS29MRCdic significantly identified induction failure with 59% sensitivity, 77% specificity, and 72% overall accuracy (odds ratio, 4.81; P = 4.15 × 10−10). PS29MRCdic was able to improve the European Leukemia Network 2017 (ELN-2017) risk classification within every category. The median overall survival with high PS29MRCdic was 1.8 years compared with 4.3 years for low-risk patients. In multivariate analysis including ELN-2017 and clinical and genetic markers, only age and PS29MRCdic were independent predictors of refractory disease. In patients aged ≥60 years, only PS29MRCdic remained as a significant variable. In summary, we confirmed PS29MRC as a valuable classifier to identify high-risk patients with AML. Risk classification can still be refined beyond ELN-2017, and predictive classifiers might facilitate clinical trials focusing on these high-risk patients with AML.

Abstract: Mutations of the isocitrate dehydrogenase-1 (IDH1) and IDH2 genes are among the most frequent alterations in acute myeloid leukemia (AML) and can be found in ∼20% of patients at diagnosis. Among 4930 patients (median age, 56 years; interquartile range, 45-66) with newly diagnosed, intensively treated AML, we identified IDH1 mutations in 423 (8.6%) and IDH2 mutations in 575 (11.7%). Overall, there were no differences in response rates or survival for patients with mutations in IDH1 or IDH2 compared with patients without mutated IDH1/2. However, distinct clinical and comutational phenotypes of the most common subtypes of IDH1/2 mutations could be associated with differences in outcome. IDH1-R132C was associated with increased age, lower white blood cell (WBC) count, less frequent comutation of NPM1 and FLT3 internal tandem mutation (ITD) as well as with lower rate of complete remission and a trend toward reduced overall survival (OS) compared with other IDH1 mutation variants and wild-type (WT) IDH1/2. In our analysis, IDH2-R172K was associated with significantly lower WBC count, more karyotype abnormalities, and less frequent comutations of NPM1 and/or FLT3-ITD. Among patients within the European LeukemiaNet 2017 intermediate- and adverse-risk groups, relapse-free survival and OS were significantly better for those with IDH2-R172K compared with WT IDH, providing evidence that AML with IDH2-R172K could be a distinct entity with a specific comutation pattern and favorable outcome. In summary, the presented data from a large cohort of patients with IDH1/2 mutated AML indicate novel and clinically relevant findings for the most common IDH mutation subtypes.

Abstract: Acute megakaryoblastic leukaemia (AMKL) is associated with poor prognosis. Limited information is available on its cytogenetics, molecular genetics and clinical outcome. We performed genetic analyses, evaluated prognostic factors and the value of allogeneic haematopoietic stem cell transplantation (allo‐HSCT) in a homogenous adult AMKL patient cohort. We retrospectively analysed 38 adult patients with AMKL (median age: 58 years, range: 21–80). Most received intensive treatment in AML Cooperative Group (AMLCG) trials between 2001 and 2016. Cytogenetic data showed an accumulation of adverse risk markers according to ELN 2017 and an unexpected high frequency of structural aberrations on chromosome arm 1q (33%). Most frequently, mutations occurred in TET2 (23%), TP53 (23%), JAK2 (19%), PTPN11 (19%) and RUNX1 (15%). Complete remission rate in 33 patients receiving intensive chemotherapy was 33% and median overall survival (OS) was 33 weeks (95% CI: 21–45). Patients undergoing allo‐HSCT ( n  = 14) had a superior median OS (68 weeks; 95% CI: 11–126) and relapse‐free survival (RFS) of 27 weeks (95% CI: 4–50), although cumulative incidence of relapse after allo‐HSCT was high (62%). The prognosis of AMKL is determined by adverse genetic risk factors and therapy resistance. So far allo‐HSCT is the only potentially curative treatment option in this dismal AML subgroup.

Abstract: Algorithmen auf Basis großer Datenmengen: Voraussetzung für die Anwendung von KI-Algorithmen ist eine hochqualitative Digitalisierung von Präparaten mit ausreichender Auflösung. Ferner ist die zuverlässige Annotation einer ausreichenden Menge von Trainings- und Testdaten nötig. Umfangreiche Bilddatenbanken für die KI-gestützte Leukämiediagnostik: Zur Entwicklung von datengetriebenen Algorithmen wie z.B. neuronaler Netze ist die Bereitstellung großer, kuratierter Datenbanken nötig. Im Bereich der morphologischen Leukozyten-Differenzierung kann dazu z.B. die Annotation von Zehntausenden von Einzelzellen nötig sein. Neuere Methoden benötigen weniger manuelle Annotationen, jedoch in der Regel noch größere Trainingsdatensätze. Methoden zur Nachvollziehbarkeit der Vorhersagen: Die „erklärbare KI“ stellt eine Reihe von Methoden bereit, die den Vorhersageprozess von neuronalen Netzen transparenter machen und für die Vorhersage relevante Anteile der Eingangsdaten erkennen. Dies ermöglicht Anwendern, die Vorhersagen des Algorithmus auf Plausibilität zu überprüfen und z.B. zu erkennen, ob bekanntermaßen diagnostisch aussagekräftige Strukturen wie Auer-Stäbchen erkannt wurden. Voraussetzungen für die Routineanwendung: Für die breite Anwendung in der diagnostischen Routine sind Stabilitäts- und Robustheitsanalysen nötig, um sicherzugehen, dass die Algorithmen ihre Leistungsfähigkeit auch bei Schwankungen der Proben- und Digitalisierungsqualität behalten. Hierzu befinden sich bei den Zulassungsinstitutionen aktuell Standards in Entwicklung. Perspektive Integrierte Diagnostik: Datenbasierte Algorithmen erlauben die Verknüpfung verschiedener diagnostischer Modalitäten und versprechen bei kombiniertem Zugang zu unterschiedlichen Befundarten eine noch höhere Genauigkeit, stellen allerdings auch noch höhere Ansprüche an die verwendete Datenbasis.

Abstract: Chemotherapy resistance is the main impediment in the treatment of acute myeloid leukaemia (AML). Despite rapid advances, the various mechanisms inducing resistance development remain to be defined in detail. Here we report that loss-of-function mutations (LOF) in the histone methyltransferase EZH2 have the potential to confer resistance against the chemotherapeutic agent cytarabine. We identify seven distinct EZH2 mutations leading to loss of H3K27 trimethylation via multiple mechanisms. Analysis of matched diagnosis and relapse samples reveal a heterogenous regulation of EZH2 and a loss of EZH2 in 50% of patients. We confirm that loss of EZH2 induces resistance against cytarabine in the cell lines HEK293T and K562 as well as in a patient-derived xenograft model. Proteomics and transcriptomics analysis reveal that resistance is conferred by upregulation of multiple direct and indirect EZH2 target genes that are involved in apoptosis evasion, augmentation of proliferation and alteration of transmembrane transporter function. Our data indicate that loss of EZH2 results in upregulation of its target genes, providing the cell with a selective growth advantage, which mediates chemotherapy resistance.